To develop neuroprotective therapies for Parkinson's disease (PD) it is necessary to achieve a better understanding of the pathogenesis of dopamine (DA) neuron degeneration. There has been tremendous growth in our understanding of both genetic and environmental causes of PD, and while there are important uniting hypotheses, convincing evidence of a single mechanism of degeneration has remained elusive. However, diverse causes of cellular injury ultimately share common pathways of programmed cell death (PCD). It therefore seems prudent, in developing neuroprotective therapies, to target these pathways. We have developed evidence in animal models that two important upstream pathways of PCD may play a role in the degeneration of DA neurons: the MAPK signaling cascade and endoplasmic reticulum (ER) stress. In relation to MAPK signaling, we have shown that c-jun phosphorylation is a universal feature of induced apoptotic death in DA neurons, and that death can be abrogated by an inhibitor of the mixed lineage kinases (MLKs), CEP11004. In our first two Aims, we will explore the molecular basis of action of this compound. We will examine the ability of dominant negative (DN) mutant forms of the MLKs to suppress death, and we will determine the effect of a DN of Akt on the protective effect of CEP11004. In Aim 3, we will examine the ability of a constitutively active form of Akt to suppress death in DA neurons. Since JNK is the principal kinase for c-jun, we hypothesize that it is a critical mediator. In the current funding period, however, we have shown in null mice that JNK3 is not necessary for c-jun phosphorylation or cell death. Therefore, in Aim 4, we will determine the isoforms essential for death to occur, using combined knock-out and DN genetic approaches. Our collaborator Dr Greene has shown that dopaminergic neurotoxins induce ER stress in vitro, and we have found that they also do so in vivo. We will therefore examine the functional role of ER stress in vivo using two approaches. In Aim 5 we will examine the sensitivity of DA neurons to neurotoxin-induced PCD in mice null for caspase-12, an important effector of ER stress-induced death. In Aim 6 we will determine the effect of overexpression of GADD34, a phosphatase for elF2alpha, on the sensitivity to induced death. We hypothesize that the inability to maintain phosphorylation of elF2alpha, which interferes with the protective components of the ER stress response, will augment sensitivity to death. The new knowledge gained from these studies will make it possible to identify precise therapeutic targets in the development of neuroprotection for DA neurons.